PHOTOSYNTHESIS (Whole Process 'in and out')

PHOTOSYNTHESIS

Photosynthesis is a vital physiological process where in the chloroplast of green plants synthesizes sugars by using water and carbon dioxide in the presence of light.  Photosynthesis literally means synthesis with the help of light i.e. plant synthesize organic matter (carbohydrates) in the presence of light.   Photosynthesis is sometimes called as carbon assimilation (assimilation: absorption into the system). This is represented by the following traditional equation.

6CO2 + 12H2O       light/green pigment                 6O2  +  C6H12O6(Carbohydrates)

During the process of photosynthesis, the light energy is converted into chemical energy and is stored in the organic matter, which is usually the carbohydrate. One molecule of glucose for instance, contains about 686 K Calories energy. CO2 and water constitute the raw material for this process and oxygen and water are formed as the by products during photosynthesis. Stephen Hales (1727) first explained the relationship between sunlight and leaves and Sachs (1887) established that starch was the visible product of photosynthesis.

Photosynthetic apparatus 

The chloroplast in green plants constitutes the photosynthetic apparatus.  In higher plants, the chloroplast is discoid in shape, 4-6 µ in length and 1-2µ thick.  The chloroplast is bounded by two unit membranes of approximately 50°A thickness and consists of lipids and proteins.  The thickness of the two membranes including the space enclosed by them is approximately 300°A (1 Angstrom: 0.1 cm).

 Internally, the chloroplast is filled with a hydrophilic matrix called as stroma embedded with grana.  Each grana consists of 5-25 disk shaped grana lamellae (thylakoid) placed one above the other like the stack of coins.  Each grana lamella of thylakoid encloses a space called loculus and the thylakoid membrane consists of alternating layer of lipids and proteins. Some of the grana lamella of thylakoid of grana are connected with thylakoid of other grana by somewhat thinner stroma lamella or fret membrane. Chlorophyll and other photosynthetic pigments are confined to grana.  The chlorophylls are the site of photochemical reactions.

 Photosynthetic pigments

 Photosynthetic pigments are of three types; Chlorophylls, Carotenoids and Phycobillins.

• Chlorophylls and Carotenoids are insoluble in water and can be extracted only with organic solvents such as acetone, petroleum ether and alcohol.

• Phycobillins are soluble in water .

• Carotenoids include carotenes and xanthophylls.  The xanthophylls are also called as carotenols.

 Chlorophylls (green pigments)

 Chlorophylls are magnesium porphyrin compounds.  The porphyrin ring consists of four pyrrol rings joined together by CH bridges. Long chain C atoms called as phytol chain is attached to porphyrin ring at pyrrol ring IV. The chemical structure of chlorophyll a and chlorophyll b are well established.  The molecular formula for chlorophyll a: C55H72O5N4 Mg and chlorophyll b: C55H70O6N4 Mg. Both of them consist of Mg porphyrin head which is hydrophilic and a phytol tail which is lipophilic.  The two chlorophylls differ because in chlorophyll b there is a –CHO group instead of CH3 group at the 3rd C atom in pyrrol ring II. Chlorophyll is formed from protochlorophyll in light.  The protochlorophyll lacks 2H atoms one each at 7th and 8th C atoms in pyrrol ring IV.

Carotenoids (yellow or orange pigments)

1. Carotenes: Carotenes are hydrocarbons with a molecular formula C40H56

2. Xanthophylls (carotenols) :

They are similar to carotenes but differ in having two oxygen atoms in the form of hydroxyl or carboxyl group.  The molecular formula is C40H56O2.The role of Carotenoids is absorption of light energy and transfer the light energy to chlorophyll a molecules. They also play a very important role in preventing photodynamic damage within the photosynthetic apparatus. Photodynamic damage is caused by O2 molecules which is very reactive and is capable of oxidizing whole range of organic compounds such as chlorophylls and there by making them unfit for their normal physiological function.

 

Phycobillins (red and blue pigments)

  These also contain four pyrrol rings but lack Mg and the phytol chain.

 

 

Mechanism of Photosynthesis 

Photo systems (Two pigment systems)

 

 The discovery of red drop and the Emerson’s enhancement effect led the scientists to suggest that photosynthesis is driven by two photochemical processes. These processes are associated with two groups of photosynthetic pigments called as pigment system I and pigment system II.  Wavelength of light shorter than 680 nm affect both the pigments systems while wavelength longer than 680 nm affect only pigment system I.

 In green plants, pigment system I contains chlorophyll a, b and carotene.  In this pigment system, a very small amount of chlorophyll a absorbing light at 700 nm, known as P700 however constitutes the reaction centre of photosystem I.

 The pigment system II contains chlorophyll b and some forms of chlorophyll a (such as chlorophyll a 662, chlorophyll a 677 and chlorophyll a 679) and xanthophylls.  A very small amount of special form of chlorophyll called P680 constitute the reaction centre of pigment system II. Carotenoids are present in both the pigment systems

 The two pigment systems I and II are interconnected by a protein complex called cytochrome b6–f complex.  The other intermediate components of electron transport chain viz., plastoquinone (PQ) and plastocyanin (PC) act as mobile electron carriers between the complex and either of the two pigment systems. The light energy absorbed by other pigment is ultimately trapped by P700 and P680 forms of chlorophyll a which alone take part in further photochemical reaction. 

 Pigment system I (PSI) complex consists of 200 chlorophylls, 50 Carotenoids and a molecule of chlorophyll a absorbing light at 700 nm(P700) and this constitute the reaction centre of photosystem I. Pigment system II (PSII) complex consists of 200 chlorophylls, 50 Carotenoids and a mole of chlorophyll a absorbing light at 680 nm, called P 680 at the centre.  This constitutes the reaction centre of pigment system II.

 Photosynthetic units – The Quantasomes 

Emerson and Arnold (1932) showed that about 2500 chlorophyll molecules are require fixing one molecule of CO2 in photosynthesis. This number of chlorophyll molecules was called the chlorophyll unit but the name was subsequently changed to photosynthetic unit.  However, since the reduction or fixation of one CO2 molecule requires about 10 quanta of light, it is assured that 10 flashes of light are required to yield one O2 molecule or reduction of one molecule of CO2.  Thus each individual unit would contain 1/10th of 2500 i.e., 250 molecules.

 

Action spectrum 

The pigments present in plants or any living organism have the ability to absorb radiant energy to carry out photo physiological reactions. It is difficult to decide which specific pigment is actually associated with the particular photochemical reactions.  Hence, a common procedure to identify the pigment involved in a particular photoreaction is to determine the action spectrum i.e. measuring the rate of the particular photoreaction. Once the action spectrum for a photo physiological reaction is determined, the next step is to compare this action spectrum with absorption spectrum of a pigment.  Two pigments, A and B were isolated from the same plant and their absorption spectra were determined.  Pigment A has a peak in absorption at 395 nm and the pigment B at 660 nm.  The close correspondence between the absorption spectrum and the action spectrum of pigment B strongly supports that Pigment B is responsible for absorbing radiant energy to drive this photoreaction.

 

Mechanism of photosynthesis 

The biosynthesis of glucose by the chloroplast of green plants using water and CO2 in the presence of light is called photosynthesis. Photosynthesis is a complex process of synthesis of organic food materials. It is a complicated oxidation- reduction process where water is oxidized and CO2 is reduced to carbohydrates. The mechanism of photosynthesis consists of two parts.

1. Light reaction / Primary photochemical reaction / Hill’s reaction/ Arnon’s cycle

 2. Dark reaction / Black man’s reaction / Path of carbon in photosynthesis.

1. Light reaction or Primary photochemical reaction or Hill’s reaction

 In light reaction, ATP and NADPH2 are produced and in the dark reaction, CO2 is reduced with the help of ATP and NADPH2 to produce glucose. The light reaction is called primary photochemical reaction as it is induced by light. Light reaction is also called as Hill’s reaction as Hill proved that chloroplast produce O2 from water in the presence of light. It is also called as Arnon’s cycle because Arnon showed that the H+ ions released by the break down of water are used to reduce the coenzyme NADP to NADPH. Light reaction includes photophosphorylation as ATP is synthesized in the presence of light.  The reaction takes place only in the presence of light in grana portion of the chloroplast and it is faster than dark reaction.  The chlorophyll absorbs the light energy and hence the chlorophyll is called as photosystem or pigment system. Chlorophylls are of different types and they absorb different wavelengths of light. Accordingly, chlorophylls exist in two photo systems, Photosystem I (PSI) and Photosystem II (PS II). Both photo systems are affected by light with wavelengths shorter than 680nm, while PS I is affected by light with wavelengths longer than 680nm.

The light reaction can be studied under the following headings.

 

 i. Absorption of light energy by chloroplast pigments 

Different chloroplast pigments absorb light in different regions of the visible part of the spectrum.

 

 

ii. Transfer of light energy from accessory pigments to chlorophyll a

All the photosynthetic pigments except chlorophyll a are called as accessory or antenna pigments.  The light energy absorbed by the accessory pigments is transferred by resonance to chlorophyll a which alone can take part in photochemical reaction.  Chlorophyll a molecule can also absorb the light energy directly. In pigment system I, the photoreaction centre is P700 and in pigment system II, it is P680.

 

iii. Activation of chlorophyll molecule by photon of light 

When P700 or P680 forms of chlorophyll a receives a photon (quantum) of light, becomes an excited molecule having more energy than the ground state energy.  After passing through the unstable second singlet state and first singlet stage the chlorophyll molecules comes to the meta stable triplet state.  This excited state of chlorophyll molecule takes part further in primary photochemical reaction i.e. the electron is expelled from the chlorophyll a molecule.

Chlorophyll a                   Excited triplet state of chlorophyll a

iv. Photolysis of water and O2 evolution (oxidation of water) 

These processes are associated with pigment system II and are catalyzed by Mn++ and Cl- ions.  When pigment system II is active i.e it receives the light, the water molecules split into OH- and H+ ions (Photolysis of water).  The OH- ions unite to form some water molecules again and release O2 and electrons.

4H2O   → 4H+ + 4 (OH-)

            4(OH-)  → 2H2O + O2 + 4e- 

 2H2O   → 4H+ + O2 + 4e

v. Electron transport and production of assimilatory powers (NADPH2 and ATP)

 It has already been observed that when chlorophyll molecule receives the photon of light, an electron is expelled from the chlorophyll a molecule along with extra energy.  This electron after traveling through a number of electron carriers is utilized for the production of NADPH2 from NADP and also utilized for the formation of ATP molecules from ADP and inorganic phosphate (Pi).  The transfer of electrons through a series of coenzymes is called electron transport and the process of formation of ATP from ADP and Pi using the energy of electron transport is called as photosynthetic phosphorylation or photophosphorylation. The types of Phosphorylation include cyclic and non- cyclic.

 

 

Cyclic electron transport and cyclic photophosphorylation

The electrons released from photosystem I goes through a series of coenzymes and returns back to the same photosystem I. This electron transport is called cyclic electron transport. The synthesis of ATP occurring in cyclic electron transport is called cyclic photophosphorylation. The cyclic electron transport involves only pigment system I.  This situation is created when the activity of pigment system II is blocked.  Under this condition,

  1. Only pigment system I remain active

 2. Photolysis of water does not take place 

3. Blockage of noncyclic ATP formation and this causes a drop in CO2 assimilation in           dark   reaction 

4. There is a consequent shortage of oxidized NADP  Thus, when P700 molecule is excited in pigment system I by absorbing a photon (quantum) of light, the ejected electron is captured by ferredoxin via FRS.  From ferredoxin, the electrons are not used up for reducing NADP to NADPH + H+ but ultimately it falls back to the P700 molecule via number of other intermediate electron carriers. The electron carriers are probably cytochrome b6, cytochrome f and plastocyanin.  During this electron transport, phosphorylation of ADP molecule to form ATP molecule take place at two places i.e., between ferredoxin and cytochrome b6 and between cytochrome b6 and cytochrome f.   Thus, two ATP molecules are produced in this cycle. Since the electron ejected from P700 molecule is cycled back, the process has been called as cyclic electron transport and the accompanying phosphorylation as the cyclic photophosphorylation.

Significance of cyclic photophosphorylation

1. During cyclic electron transport and phosphorylation, photolysis of water, O2 evolution and reduction of NADP do not take place.

2. The electron returns or cycles back to original position in the P700 form of chlorophyll a. Here, chlorophyll molecule serves both as donor and acceptor of the electron.

 3. It generates energy rich ATP molecules at two sites and as such cannot drive dark reactions of photosynthesis 

On the other hand, non- cyclic photophosphorylation does not produce sufficient ATP in relation to NADPH to operate the dark phase of photosynthesis.  Therefore, the deficiency of ATP molecule in non–cyclic photophosphorylation is made up by the operations of cyclic photophosphorylation.  Secondly, the cyclic photophosphorylation may be an important process in providing ATP for photosynthesis and other processes such as synthesis of starch, proteins, lipids, nucleic acids and pigments within the chloroplast. Non cyclic photophosphorylation   The electron released from photosystem II goes through a series of enzymes and Coenzymes to photosystem I.   This is called non cyclic electron transport and the Synthesis of ATP in non cyclic electron transport is called non- cyclic photo phosphorylation. The main function of non cyclic electron transport is to produce the assimilatory powers such as NADPH2 and ATP and the process occurs in photosystem I and II.  This process of electron transport is initiated by the absorption of a photon (quantum) of light by P680 form of chlorophyll a molecule in the pigment system II, which gets excited and an electron is ejected from it so that an electron deficiency or a hole is left behind in the P680 molecule.  The ejected electron is trapped by an unknown compound known as Q.  From Q, the electron passes downhill along a series of compounds or intermediated electron carriers such as cytochrome b6, plastoquinone, cytochrome f and a copper containing plastocyanin and ultimately received by pigment system I.  At one place during electron transport i.e. between plastoquinone and cytochrome f, one molecule of ATP is formed from ADP and inorganic phosphate.  Now, when a photon of light is absorbed by P700 form of chlorophyll molecule in the pigment system I, this gets excited and an electron is ejected from it.  This ejected electron is trapped by FRS (Ferredoxin Reducing Substance) and it is then transferred to a non-heme iron protein called ferredoxin.  From ferredoxin, electron is transferred to NADP so that NADP is reduced to NADPH + H+  The hole in pigment system I has been filled by electron coming from pigment system II.  But, the hole or an electron deficiency in pigment system II is filled up by the electron coming from photolysis of water where, water acts as electron donor.  In this scheme of electron transport, the electron ejected from pigment system II did not return to its place of origin, instead it is taken up by pigment system I.  Similarly, the electron ejected from pigment system I did not cycle back and was consumed in reducing NADP.  Therefore, this electron transport has been called as non–cyclic electron transport and accompanying phosphorylation as non–cyclic photophosphorylation.  The non cyclic electron transport (photophosphorylation) takes the shape of Z and hence it is called by the name Z–scheme. Non cyclic photophosphorylation and O2 evolution are inhibited by CMU (3-(4’-Chlorophyl) – 1-1dimethyl urea and 3-(3-4-dichlorophenyl)-1, 1-dimethyl urea (DCMU).

 

Significance of non cyclic electron transport

1. It involves PS I and PSII

2. The electron expelled from P680 of PSII is transferred to PS I and hence it is a non cyclic electron transport. 

3. In non cyclic electron transport, photolysis of water (Hill’s reaction and evolution of O2) takes place. 

4. Phosphorylation (synthesis of ATP molecules) takes place at only one place.

5. The electron released during photolysis of water is transferred to PS II.

6. The hydrogen ions (H+) released from water are accepted by NADP and it becomes NADPH2

7. At the end of non cyclic electron transport, energy rich ATP, assimilatory power NADPH2 and oxygen from photolysis of water are observed.

 8. The ATP and NADPH2 are essential for the dark reaction wherein, reduction of CO2 to carbohydrate takes place.

Significance of light reaction

1. Light reaction takes place in chlorophyll in the presence of light.

 2. During light reaction, the assimilatory powers ATP and NADPH2 are synthesized.

3. The assimilatory powers are used in dark reaction for the conversion of CO2 into sugars.

4. Photolysis of water occurs in light reaction. The H+ ions released from water are used for the synthesis of NADPH2

5. Plants release O2 during light reaction

Red drop and Emerson’s enhancement effect 

Robert Emerson noticed a sharp decrease in quantum yield at wavelength greater than 680 nm, while determining the quantum yield of photosynthesis in chlorella using monochromatic light of different wavelengths. Since this decrease in quantum yield took place in the red part of the spectrum, the phenomenon was called as red drop.   Later, they found that the inefficient far-red light beyond 680 nm could be made fully efficient if supplemented with light of shorter wavelength (blue light).  The quantum yield from the two combined beams of light was found to be greater than the sum effects of both beams used separately. This enhancement of photosynthesis is called as Emerson’s Enhancement.

 

Dark reaction or Blackman’s reaction or Path of carbon in photosynthesis

This is the second step in the mechanism of photosynthesis. The chemical processes of photosynthesis occurring independent of light is called dark reaction. It takes place in the stroma of chloroplast. The dark reaction is purely enzymatic and it is slower than the light reaction. The dark reactions occur also in the presence of light. In dark reaction, the sugars are synthesized from CO2. The energy poor CO2 is fixed to energy rich carbohydrates using the energy rich compound, ATP and the assimilatory power, NADPH2 of light reaction. The process is called carbon fixation or carbon assimilation. Since Blackman demonstrated the existence of dark reaction, the reaction is also called as Blackman’s reaction. In dark reaction two types of cyclic reactions occur

1. Calvin cycle or C3 cycle

2. Hatch and Slack pathway or C4 cycle

Calvin cycle or C3 cycle 

It is a cyclic reaction occurring in the dark phase of photosynthesis. In this reaction, CO2 is converted into sugars and hence it is a process of carbon fixation. The Calvin cycle was first observed by Melvin Calvin in chlorella, unicellular green algae. Calvin was awarded Nobel Prize for this work in 1961. Since the first stable compound in Calvin cycle is a 3 carbon compound (3 phosphoglyceric acid), the cycle is also called as C3 cycle. The reactions of Calvin’s cycle occur in three phases.

1. Carboxylative phase

2. Reductive phase

 3. Regenerative phase

 

1. Carboxylative phase 

Three molecules of CO2 are accepted by 3 molecules of 5C compound viz., ribulose diphosphate to form three molecules of an unstable intermediate 6C compound. This reaction is catalyzed by the enzyme, carboxy dismutase

3 CO2 + 3 Ribulose diphosphate Carboxy dismutase 3 unstable intermediate 6 carbon compound

The three molecules of the unstable 6 carbon compound are converted by the addition of 3 molecules of water into six molecules of 3 phosphoglyceric acid. This reaction is also catalyzed by the enzyme carboxy mutase.

3 unstable intermediate 6 C compound + 3 H2O Carboxy dismutase 3 phosphoglyceric acid

3 phosphoglyceric acid (PGA) is the first stable product of dark reaction of photosynthesis and since it is a 3 carbon compound, this cycle is known as C3 cycle

 

2. Reductive phase

 Six molecules of 3PGA are phosphorylated by 6 molecules of ATP (produced in the light reaction) to yield 6 molecules of 1-3 diphospho glyceric acid and 6 molecules of ADP. This reaction is catalyzed by the enzyme, Kinase

3 Phospho  glyceric acid + ATP   Kinase 1,3 diphospho glyceric acid + ADP

Six molecules of 1, 3 diphosphoglyceric acid are reduced with the use of 6 molecules of NADPH2 (produced in light reaction) to form 6 molecules of 3 phospho glyceraldehyde. This reaction is catalysed by the enzyme, triose phosphate dehydrogenase.

1,3 diphospho glyceric acid + NADPH2 Triose phosphate  Dehydrogenase 3 phospho glyceraldehyde + NADP   + H3PO4

 

3. Regenerative phase 

In the regenerative phase, the ribose diphosphate is regenerated. The regenerative phase is called as pentose phosphate pathway or hexose monophophate shunt. It involves the following steps.

1. Some of the molecules of 3 phospho glyceraldehyde into dihydroxy acetone phosphate. Both 3 phospho glyceraldehyde and dihydroxy acetone phosphate then unite in the presence of the enzyme, aldolase to form fructose, 1-6 diphosphate.

3 phospho glyceraldehyde  Triose phosphate  isomerase       Dihydroxy acetone PO4        (DHAP)

3 phospho    glyceraldehyde + DHAP Aldolase Fructose 1,6 diphosphate

 

2. Fructose 6 phosphate is converted into fructose 6 phosphate in the presence of phosphorylase

fructose 1,6 diphosphate Phosphorylase Fructose 6 phosphate

3. Some of the molecules of 3 phospho glyceraldehyde instead of forming hexose sugars are diverted to regenerate ribulose 1-5 diphosphate

3 phospho glyceraldehyde         Ribulose 1,5 diphosphate

4. 3 phospho glyceraldehyde reacts with fructose 6 phosphate in the presence of enzyme transketolase to form erythrose 4 phosphate ( 4C sugar) and xylulose 5 phosphate(5C sugar)

3 phospho glyceraldehyde +Fructose 6 phosphate Transketolase Erythrose 4 phosphate  + Xylulose 5 phosphate

5. Erythrose 4 phosphate combines with dihydroxy acetone phosphate in the presence of the enzyme aldolase to form sedoheptulose 1,7 diphosphate(7C sugar)

Erythrose 4 phosphate   + DHAP     Aldolase     Sedoheptulose 1 ,7  diphosphate

6. Sedoheptulose 1, 7 diphosphate loses one phosphate group in the presence of the enzyme phosphatase to form sedoheptulose 7 phosphate.

Sedoheptulose 1 ,7     diphosphate + ADP  Phosphatase Sedoheptulose 7 phosphate +  ATP

7. Sedoheptulose  phosphate reacts with 3 phospho glyceraldehyde in the presence of  transketolase to form xylulose 5 phosphate and ribose 5 phosphate ( both % c sugars)

Sedoheptulose  7 phosphate + 3 phospho     glyceraldehyde Transketolase Xylulose  5 phosphate       Ribose  5     +   phosphate

 

8. Ribose 5 phosphate is converted into ribulose 1, 5 diphosphate in the presence of enzyme, phosphopentose kinase and ATP. Two molecules of xylulose phosphate are also converted into one molecule of ribulose monophosphate. The ribulose monophosphate is phosphorylated by ATP to form ribulose diphosphate and ADP, thus completing Calvin cycle.

Ribose   5 phosphate + ATP Phophopentokinase Ribulose 1,5 diphosphate +  ADP 

2 mols of  Xylulose        phosphate  Phophopentokinase 1 mol of Ribulose mono    phosphate

In the dark reaction, CO2 is fixed to carbohydrates and the CO2 acceptor ribulose diphosphate is regenerated. In Calvin cycle, 12 NADPH2 and 18 ATPs are required to fix 6 CO2 molecules into one hexose sugar molecule (fructose 6 phosphate).

6 CO2  +   12 NADPH2 + 18 ATP         Fructose 6 phosphate   +  12 NADP+  18 ADP+  17 Pi

 

 

C4 cycle or Hatch and Slack pathway

 It is the alternate pathway of C3 cycle to fix CO2.  In this cycle, the first formed stable compound is a 4 carbon compound viz., oxaloacetic acid. Hence it is called C4 cycle. The path way is also called as Hatch and Slack as they worked out the pathway in 1966 and it is also called as C4 dicarboxylic acid pathway. This pathway is commonly seen in many grasses, sugar cane, maize, sorghum and amaranthus.  The C4 plants show a different type of leaf anatomy. The chloroplasts are dimorphic in nature. In the leaves of these plants, the vascular bundles are surrounded by bundle sheath of larger parenchymatous cells. These bundle sheath cells have chloroplasts. These chloroplasts of bundle sheath are larger, lack grana and contain starch grains. The chloroplasts in mesophyll cells are smaller and always contain grana. This peculiar anatomy of leaves of C4 plants is called Kranz anatomy. The bundle sheath cells are bigger and look like a ring or wreath. Kranz in German means wreath and hence it is called Kranz anatomy. The C4 cycle involves two carboxylation reactions, one taking place in chloroplasts of mesophyll cells and another in chloroplasts of bundle sheath cells. There are four steps in Hatch and Slack cycle: 1. Carboxylation

 2. Breakdown

3. Splitting

4. Phosphorylation

1. Carboxylation 

It takes place in the chloroplasts of mesophyll cells. Phosphoenolpyruvate, a 3 carbon compound picks up CO2 and changes into 4 carbon oxaloacetate in the presence of water. This reaction is catalysed by the enzyme, phosphoenol pyruvate carboxylase.

2. Breakdown

 Oxaloacetate breaks down readily into 4 carbon malate and aspartate in the presence of the enzyme, transaminase and malate dehydrogenase.

3. Splitting  

In the sheath cells, malate and aspartate split enzymatically to yield free CO2 and 3 carbon pyruvate. The CO2 is used in Calvin’s cycle in the sheath cell.  The second Carboxylation occurs in the chloroplast of bundle sheath cells. The CO2 is accepted by 5 carbon compound ribulose diphosphate in the presence of the enzyme, carboxy dismutase and ultimately yields 3 phosphoglyceric acid. Some of the 3 phosphoglyceric acid is utilized in the formation of sugars and the rest regenerate ribulose diphosphate.  

 4. Phosphorylation 

The pyruvate molecule is transferred to chloroplasts of mesophyll cells where, it is phosphorylated to regenerate phosphoenol pyruvate in the presence of ATP. This reaction is catalysed by pyruvate phosphokinase and the phophoenol pyruvate is regenerated.

            In Hatch and Slack pathway, the C3 and C4 cycles of carboxylation are linked and this is due to the Kranz anatomy of the leaves. The C4 plants are more efficient in photosynthesis than the C3 plants. The enzyme, phosphoenol pyruvate carboxylase of the C4 cycle is found to have more affinity for CO2 than the ribulose diphosphate carboxylase of the C3 cycle in fixing the molecular CO2 in organic compound during Carboxylation.

Crassulacean Acid Metabolism (CAM) cycle or the dark fixation of CO2 in succulents 

CAM is a cyclic reaction occurring in the dark phase of photosynthesis in the plants of Crassulaceae. It is a CO2 fixation process wherein, the first product is malic acid. It is the third alternate pathway of Calvin cycle, occurring in mesophyll cells. The plants exhibiting CAM cycle are called CAM plants. Most of the CAM plants are succulents e.g., Bryophyllum, Kalanchoe, Crassula, Sedium, Kleinia etc. It is also seen in certain plants of Cactus e.g. Opuntia, Orchid and Pine apple families.  

CAM plants are usually succulents and they grow under extremely xeric conditions. In these plants, the leaves are succulent or fleshy. The mesophyll cells have larger number of chloroplasts and the vascular bundles are not surrounded by well defined bundle sheath cells.  In these plants, the stomata remain open during night and closed during day time. The CAM plants are adapted to photosynthesis and survival under adverse xeric conditions. CAM plants are not as efficient as C4 plants in photosynthesis. But they are better suited to conditions of extreme desiccation. CAM involves two steps:

1. Acidification

2. Deacidification

 Factors affecting photosynthesis

 I. External factors

1. Light

 It is the most important factor of photosynthesis. Any kind of artificial light such as electric light can induce photosynthesis. Out of the total solar energy, only 1-2 % is used for photosynthesis and the rest is used for other metabolic activities. The effect of light on photosynthesis can be studied under three categories.

2. Carbon dioxide 

CO2 is one of the raw materials required for photosynthesis. If the CO2 concentration is increased at optimum temperature and light intensity, the rate of photosynthesis increases. But, it is also reported that very high concentration of CO2 is toxic to plants inhibiting photosynthesis

3. Temperature   

The rate of photosynthesis increases by increase in temperature up to 40 ºC and after this, there is reduction in photosynthesis. High temperature results in the denaturation of enzymes and thus, the dark reaction is affected. The temperature requirement for optimum photosynthesis varies with the plant species. For example, photosynthesis stops in many plants at 0 ºC but in some conifers, it can occur even at -35 ºC. Similarly photosynthesis stops beyond 40-50 ºC in certain plants; but certain bacteria and blue green algae can perform photosynthesis even at 70 ºC.

4. Water 

Water has indirect effect on the rate of photosynthesis although it is one of the raw materials for the process. The amount of water utilized in photosynthesis is quite small and even less than 1 per cent of the water absorbed by a plant. Water rarely acts as a limiting factor for photosynthesis. During water scarcity, the cells become flaccid and the rate of photosynthesis might go down.

 

 

5. Oxygen 

Oxygen is a byproduct of photosynthesis and an increase in the O2 concentration in many plants results in a decrease in the rate of photosynthesis. The phenomenon of inhibition of photosynthesis by o2 was first discovered by Warburg (1920) in green alga Chlorella and this effect is known as Warburg’s effect. This is commonly observed in C3 plants. 

 In plants, there is a close relationship between Warburg’s effect and photorespiration. The substrate of photorespiration is glycolate and it is synthesized from some intermediates of Calvin’s cycle. In plants that show Warburg’s effect, increased O2 concentration result in diversion of these intermediates of Calvin cycle into the synthesis of glycolate, thereby showing higher rate of photorespiration and lower photosynthetic productivity.

6. Mineral elements 

The elements like Mg. Fe, Cu, Cl, Mn, P etc are involved in the key reactions of photosynthesis and hence, the deficiency of any of these nutrients caused reduction in photosynthesis

7. Chlorophyll content

 It is very much essential to tarp the light energy. In 1929, Emerson found direct relationship between the chlorophyll content and rate of photosynthesis. In general, the chlorophyll sufficient plants are green in colour showing efficient photosynthesis. The chlorotic leaves due to irregular synthesis of chlorophyll or breakdown of chlorophyll pigment exhibit inefficient photosynthesis.

8. Leaf 

The leaf characters such as leaf size, chlorophyll content, number of stomata. Leaf orientation and leaf age are some of the factors that are responsible for photosynthesis. The maximum photosynthetic activity is usually seen in the physiologically functional and full size leaves (usually third/fourth leaf from the tip of the shoot system).

9. Carbohydrates

  If the accumulated carbohydrates are not translocated, the photosynthetic rate is reduced and respiration is increased. Sugar is converted into starch and gets accumulated in the chloroplasts. This reduces the effective surface in the chloroplast and the rate of photosynthesis is decreased.

10. Phytohormones

 Treharne (1970) reported first that photosynthesis may be regulated by plant hormone system. He found that gibberellic acid and cytokinin increase the carboxylating activity and photosynthetic rates. Meidner (1967) also reported that kinetin @ 3µm causes 12 per cent increase in photosynthesis within one hour of the treatment.